Extended RNA synthesis in isolated nuclei from rat pituitary tumor cells

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Abstract
Nuclei of GH3 cells, isolated by detergent lysis, synthesized RNA for an extended period at 29.degree. C in the presence of rat liver ribonuclease inhibitor (RI). Extended RNA synthesis was dependent upon the presence of RI. Sucrose gradient sedimentation analysis of the cell-free reaction products showed that RNA ranging from 4 S to > 28 S were synthesized. Further characterization of the RNA products was made by examining the sensitivity of synthesis to .alpha.-amanitin and actinomycin D as well as by oligo(dT)-cellulose binding properties. RNA polymerases I, II and III were functioning in isolated GH3 nuclei. Newly synthesized RNA were found in both the nuclear pellet and postnuclear supernatant fractions. RNA polymerase I products remained associated with the nuclear pellet throughout a 60-min incubation period; RNA synthesized by RNA polymerase III emerged rapidly into the supernatant fraction. RNA polymerase II products were distributed in both fractions and contained poly(A). De novo poly(A) synthesis was demonstrated and was inhibited by cordycepin triphosphate (3''-dATP). Supernatant RNA synthesized by polymerase II contained a poly(A) segment of about 150 adenine residues; these transcripts sedimented heterogeneously with an apparent size distribution (under denaturing conditions) which was smaller than that of nuclear RNA polymerase II products and resembled that of cellular mRNA. Qualitative differences in the nuclear and supernatant RNA, the kinetics of appearance of the latter and the differential effect of 3''-dATP on the extranuclear appearance of supernatant RNA suggest that a process resembling nuclear-cytoplasmic RNA transport occurred in this cell-free nuclear system.